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Graphene oxide (GO) is a promising material, which is likely to find applications in the fields of medicine and biotechnology. However, the current knowledge of its influence on human organism is limited. Even less information is available on the effects of GO on the cell lines widely used in biotechnology. The aim of this work is to describe the interaction between GO nanoparticles and BAP3 hybridoma cells which produce anti-human-PSG1 IgG, in vitro. We studied the effect of GO nanoparticles on cell viability and the intensity of internalization (adhesion) of nanoparticles by the cells. We used GO nanoparticles of different size, with surface being functionalized by linear or branched PEG (GO-PEG). The PEG coating level was 20% (by mass). The following nanoparticle concentrations were used: 5 g/mL and 25 g/mL. The BAP3 cells were cultured in a 48-well cell culture plates in serum-free DCCM-1 media in the presence of GO nanoparticles. The cells were cultured for 24 hours at 37 С and 5% СО2. Cell viability was assessed by a flow cytometer utilizing Zombie Aqua (ZA) staining. Internalization (adhesion) of nanoparticles was monitored using a flow cytometer by GO fluorescense in the samples (ex = 488 nm). Moreover, interactions between hybridoma cells and GO nanoparticles were visualized by EVOS M5000 visualization system, which included an inverted fluorescent microscope. We demonstrated that GO nanoparticles possess a cytotoxic effect when applied at high concentration (25 g/mL). The highest cytotoxic effect is caused by GO nanoparticles coated with linear PEG. The degree of nanoparticle internalization (adhesion) was shown to be significantly lower when the particles were present at lower (5 g/mL) concentration. Internalization (adhesion) of nanoparticles of smaller size was more abundant. Furthermore, these nanoparticles were shown to have a stronger cytotoxic effect compared to larger particles. In general, cytotoxicity of GO nanoparticles decreases with increasing size, which is especially evident if the fact that the mean effective diameter of the nanoparticles coated with branched PEG is considered larger than their linear PEG-coated counterparts. The data obtained allow us to draw a correlation between the cytotoxic effect of GO nanoparticles and the level of their internalization (adhesion) by the cells. In general, this work concerns some novel aspects of interaction between GO nanoparticles and hybridoma cells.

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation.

The embryo, being half an antigenically “foreign” organism, should elicit a maternal immune response. During evolution, however, the mechanisms ensuring successful development of pregnancy have been formed. In particular, among factors providing immune tolerance during pregnancy are some proteins associated with pregnancy. The pregnancy-specific β 1-glycoprotein (PSG, PSG1; SP1; PSβG1) is a dominant fetoplacental protein produced by cyto- and syncytiotrophoblast cells, and it exhibits immunosuppressive properties. Our team of authors possesses a patented method for obtaining native human PSG preparation from blood serum of pregnant women, a mixture of PSG1, PSG3, PSG7, PSG9, and their isoforms and precursors. This review presents an analysis of our results for the period from 2015 to 2020. We studied the immunoregu-latory effects of the obtained PSG preparation at concentrations comparable to those observed in pregnancy (1, 10, 100 |ag/mL). The study was performed with peripheral blood cells obtained from non-pregnant women. It was found that PSG significantly increased the percentage of adaptive Tregs in vitro, as well as expression of CTLA-4, GITR, and production of IL-10 by these cells. It has been shown that PSG has a stimulating effect upon indoleamine-2,3-dioxygenase (IDO) activity of peripheral blood monocytes. For Th17 cells, we have demonstrated that PSG can suppress differentiation and proliferation of these cells, along with reduced production of critical proinflammatory cytokines (IL-8, IL-10, IL-17, IFNγ, MCP-1, TNF α). As for the memory T cells, PSG suppressed CD25 expression and IL-2 production by them, along with simultaneous decreased expression of Gfi1, hnRNPLL genes, thus preventing the formation of the “mature” CD45R0 isoform. PSG has been shown to inhibit naive T cells’ conversion to the terminally differentiated effector subpopulation of helper T cells. When analyzing PSG effects upon cytokine profile of immunocompetent cells, it was found that the protein predominantly suppresses the Th1 cytokine production by the studied cell types, and regulates the Th2 cytokine production in divergent manner. The results obtained are consistent with general concept of immunosuppression during pregnancy. Thus, PSG could be one of the factors preventing formation and implementation of immune response to placental antigens.

A nuclear magnetic resonance (NMR) immunoassay based on the application of carbon-coated iron nanoparticles conjugated with recognition molecules was designed. The principle of the assay is that ELISA plates are coated with a capture element, and then an analyte is added and detected by conjugating the magnetic nanoparticles with recognition molecules. Afterwards, the elution solution (0.1-M sodium hydroxide) is added to displace the magnetic nanoparticles from the well surfaces into the solution. The detached magnetic nanoparticles reduce transverse relaxation time (T2) values of protons from the surrounding solution. A portable NMR relaxometer is used to measure the T2. Magnetic nanoparticles conjugated with streptavidin, monoclonal antibodies, and protein G were applied for the detection of biotinylated albumin, prostate-specific antigen, and IgG specific to tetanus toxoid (TT). The limit of detection of anti-TT IgG was 0.08–0.12 mIU/mL. The reproducibility of the assay was within the acceptable range (CV

We studied the effects of pregnancy-specific β1-glycoprotein (PSG) on the replicative potential of naive T cells (CD45RA+) and immune memory T cells (CD45R0+) in vitro by evaluating the expression of the hTERT gene in combination with the proliferative activity of cells. Human PSG was obtained by the author's patented method of immunopurification using a biospecific sorbent with subsequent removal of immunoglobulin contamination on a HiTrap Protein G HP column. We used monocultures of CD45RA+ and CD45R0+ lymphocytes isolated from peripheral blood mononuclear cells of reproductive-age women. It was found that PSG in physiological concentrations inhibited the expression of the hTERT gene mRNA in naive T cells and immune memory T cells and simultaneously reduced the number of proliferating T cells estimated by the differential gating method. At the same time, PSG reduced CD71 expression only on naive T cells without affecting this molecule on immune memory T cells. Thus, PSG decreased the replication potential and suppressed the proliferation of T cells and immune memory T cells, which in the context of pregnancy can contribute to the formation of immune tolerance to the semi-allogeneic embryo.

Graphene-based preparations are the most promising materials in biomedicine. This review is aimed at analyzing data on the interaction of graphene oxide nanoparticles with different types of cells of the immune system: neutrophils, monocytes, macrophages, dendritic cells, T- and B-lymphocytes, NK and iNKT cells. Scopus publications from 2011 to May 2020 were analyzed. The primary vector of the graphene oxide nanoparticles' effects is associated with cell activation and the formation of a proinflamma-tory profile of the immune response. At the same time, the functionalization of the graphene oxide surface with the biocompatible polymers leads to a decrease in its cytotoxicity, and in some cases, to suppression of cell activation. The interaction of graphene oxide nanoparticles with cells depends on numerous factors, such as direct and lateral sizes, oxidation state, functionalization, number of layers, 3D configuration, as well as the microbiological purity and pyrogenicity of graphene. Together, these characteristics determine whether graphene oxide nanoparticles must stimulate or suppress the immune system. These multidirectional possibilities of graphene oxide can be useful in the development of adjuvants, new drug delivery mechanisms, and modern biosensors.

Myeloid suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that generally differentiate into macrophages, granulocytes, and dendritic cells. However, in pathology, these cells acquire a suppressor phenotype, blocking immune response. In particular, MDSC levels increase in many pathological conditions, including inflammation, sepsis, traumatic shock, autoimmune diseases, cancer, and pregnancy. Over the past 12 years, an interest in examining this cell population has been steadily increased [PUBMED: 2008 (65 articles); 2020 ( 650 entries)] that will expand our understanding of immune system functioning. In humans, MDSCs are characterized by HLA-DR-CD33+CD11b+ phenotype, in turn being subdivided into CD15+ or CD66+ granulocytic (G-MDSC), CD14+ monocytic (M-MDSC), and early (e-MDSC) MDSC bearing HLA-DR-CD11b+CD33+CD14-CD66b- phenotype. This work was aimed to develop an adequate experimental model allowing to evaluate cytokine-driven differentiation of human MDSCs from peripheral blood mononuclear cells in long-term in vitro culture system. For this, peripheral blood mononuclear cells were isolated from healthy donors induced to express MDSC phenotype with GM-CSF and IL-6 (40 or 20 ng/ml) cultured for 7, 14, 21 days. In several experiments, LPS (100 ng/ml) was added to the cultured cells 24 hours before immunophenotyping. The percentage of living Zombie Aquanegative cells in cultures (gated on cells according to FSC/SSC) ranged from 90.5-93.9%. No significant differences were observed between cultured cells. In our experimental conditions, the mean percentage of total MDSC subpopulation reached 2-2.3% of total living cells, exceeding that one by 9-10-fold found in freshly isolated mononuclear cells from healthy subjects. Based on the results of our experimental study, we found that induction of e-MDSC derived from human peripheral blood mononuclear cells requires two weeks of co-culture with 40 ng/ml IL-6 and 40 ng/ml GM-CSF. “Mature” MDSCs (M-MDSC + G-MDSC) yield was peaked in the following conditions: co-culture for 3 weeks with 20 ng/ml IL-6 and 20 ng/ml GM-CSF added with 100 ng/ml LPS 24 hours before completing protocol. Overall, further examining factors modulating MDSC differentiation will reveal conditions necessary for generating this suppressor cell subset potentially used in clinical practice.

Contemporary immunoassays commonly used in clinical diagnostics mostly utilize enzymes, such as horseradish peroxidase, for signal generation. Numerous research is dedicated to the development of artificial peroxidase-mimicking catalysts with lower cost, high activity, better operational stability, and tunable properties. Herein we synthesized hemin-loaded bovine serum albumin (BSA) nanoparticles and applied them as catalytic labels (nanozymes) in colorimetric immunoassay of anti-tetanus antibodies. Hemin is a key part of the peroxidase catalytic center, possessing peroxidase like-activity. Albumin nanoparticles were loaded with multiple hemin molecules and decorated with Streptococcal protein G. Resulting nanozymes possessed good colloidal stability and allowed for antibody detection in blood serum. The sensitivity of antibody detection was sufficient for the assessment of post-vaccination immunity.

From the point of view of reproductive immunology, pregnancy is the only physiological phenomenon in which the immune system, recognizing fetal alloantigens, implements a response in the form of systemic (central) and peripheral tolerance. The factors that induce central tolerance in pregnancy are proteins associated with pregnancy. Conceptually, the embryo is a semi-allogenic transplant. Presumably, only a few molecules regulate the immune tolerance of the mother - glycodelin, human chorionic gonadotropin (hCG), Pregnancy-specific β1-glycoprotein (PSG), and alpha-fetoprotein (AFP) can be included in this list. Significantly, these molecules have evolved to perform a range of biological functions, including immunosuppression. Logically, it is necessary to try to apply the evolutionary potential in the practice of therapy for autoimmune diseases and transplantation. In this review, we consider information about the mechanisms of protein’s action on immune tolerance, as well as options for their using as pharmacological drugs. Exactly clinical situations require suppression “point” of the immune response.

The review presents data on the role of glycodelin A (GdA, PP14, α2-PEG, EP15, PEP, AUP, PAEP) in regulating the functions of the immune system in the context of the formation of feto-maternal immune tolerance during pregnancy. Glycodelin was first isolated and identified in 1976 by D.D. Petrunin. and Tatarinov Yu.S. with colleagues as a new placenta antigen, which was named chorionic α2-microglobulin. Since then, a huge amount of scientific data has been obtained on the structure, properties, and biological effects of this glycoprotein. This protein has four differentially glycosylated isoforms, namely GdA, GdF, GdC, and GdS, which are secreted in different parts of the reproductive system. The most studied isoform, glycodelin A (GdA), is secreted by the decidual glandular epithelium and accumulates in the amniotic fluid and maternal serum during pregnancy. GdA level is a sign of endometrial fertile function. GdA has diverse biological effects, in particular, it modulates endocrine function and differentiation of trophoblast cells. The role of GdA in the regulation of the immune system is to inhibit the proliferation of T and B lymphocytes, suppress the cytotoxicity of NK cells, induce apoptosis of activated CD4+ cells, monocytes and NK cells, inhibit the activity of cytotoxic T lymphocytes and suppress the functional activity of macrophages and dendritic cells. In addition, GdA increases the level of regulatory T cells, regulates the Th1/Th2 balance towards Th2, and induces a tolerant phenotype of dendritic cells. The immunomodulating activity of GdA depends on the degree of its glycosylation, which, in turn, is associated with the preparation obtaining method. Therefore, the review analyzed the features of the immunomodulating effects of the native and recombinant types of glycodelin on the immune system cells. However, the cumulative effects of GdA on the cells of the immune system make it possible to consider it as one of the main factors shaping the feto-maternal immune tolerance during pregnancy. It is also important to note that clinical studies have revealed a correlation between low levels of circulating GdA and repetitive spontaneous abortions that confirms the importance of this protein in fetoprotection. In general, it is obvious that GdA has the potential as a medicinal preparation for autoimmune deseases treatment, post-transplant complications and in vitro “reprogramming” of autoreactive T-cell clones for further cellular immunotherapy.

We studied the role of native α-fetoprotein preparation in the regulation of proliferation and functional activity of naive T cells and immune memory T cells in vitro. The study was carried out on separated fractions of naive T cells (CD45RA+) and immune memory T cells (CD45R0+) incubated with α-fetoprotein under conditions of TCR activation. At the level of naive T cells, α-fetoprotein in a concentration of 100 U/ml reduced the expression of CD28, but increased the expression of CD25, while at the level of immune memory T cells α-fetoprotein (50 and 100 U/ml) only suppressed the expression of CD25. No effects of α-fetoprotein on the proliferative status of the studied lymphocyte subpopulations and on the expression of CD71 (proliferation marker) by these cells were detected. Addition of α-fetoprotein in a concentration of 100 U/ml increased the level of IL-2 in naive T cell culture supernatants, while production of IL-2 by memory T cells remained unchanged. These data demonstrated the priority aspects of regulation of the functional activities of naive T cells and immune memory T cells.

In this work, we developed and optimized conjugates of carbon-coated iron nanoparticles (Fe@C) with streptavidin and monoclonal antibodies. The conjugation procedure included two stages. First, amino groups were grafted onto the carbon shell to facilitate noncovalent sorption of bovine serum albumin (BSA). Further, the covalent attachment of proteins to the BSA layer via glutaraldehyde coupling was performed. It was established and confirmed that the synthesis procedure is reproducible and allows preparation of stable conjugates. The resulting nanoparticles are clusters of Fe@C particles coated by proteins. The size of the clusters is in the range of 100–190 nm and can be controlled via the tuning of conjugation conditions, including pH, BSA-to-Fe@C ratio, etc. Conjugates of Fe@C with streptavidin and monoclonal antibodies (sizes of approximately 140–150 nm) were synthesized. Proton T2 relaxometry was used to detect these conjugates with very high sensitivity due to the magnetic markers, Fe@C. The relaxivity (r2) of different conjugates varied within the range of 290–450 1/s*mM. Conjugate applicability for relaxometry-based assay was confirmed by direct detection of streptococcal protein G and biotinylated BSA in a dot immunoassay.

We studied the effect of graphene oxide nanoparticles on the differentiation of human dendritic cells and uptake of nanoparticles by these cells in vitro. The objects of the study were mononuclear cells from healthy donors induced into the phenotype of dendritic cells by cytokines (IL-6 and GM-CSF). We used graphene oxide nanoparticles of different sizes functionalized with linear or branched PEG (P-GO or bP-GO) in concentrations of 5 and 25 μg/ml. It was found that graphene oxide nanoparticles did not affect the viability and percentage of dendritic cells in the culture. However, P-GO nanoparticles (25 μg/ml) suppressed the expression of CD83 on the surface of dendritic cells in cultures, thereby suppressing cell differentiation. Dendritic cells internalized P-GO nanoparticles, particles in high concentration were more actively engulfed, but the size of the particles and the type of PEG did not affect the intensity of this process. In general, P-GO nanoparticles in a concentration of 25 μg/ml can regulate differentiation of dendritic cells by suppressing their maturation.

Gelatin nanoparticles found numerous applications in drug delivery, bioimaging, immunotherapy, and vaccine development as well as in biotechnology and food science. Synthesis of gelatin nanoparticles is usually made by a two-step desolvation method, which, despite providing stable and homogeneous nanoparticles, has many limitations, namely complex procedure, low yields, and poor reproducibility of the first desolvation step. Herein, we present a modified one-step desolvation method, which enables the quick, simple, and reproducible synthesis of gelatin nanoparticles. Using the proposed method one can prepare gelatin nanoparticles from any type of gelatin with any bloom number, even with the lowest ones, which remains unattainable for the traditional two-step technique. The method relies on quick one-time addition of poor solvent (preferably isopropyl alcohol) to gelatin solution in the absence of stirring. We applied the modified desolvation method to synthesize nanoparticles from porcine, bovine, and fish gelatin with bloom values from 62 to 225 on the hundreds-of-milligram scale. Synthesized nanoparticles had average diameters between 130 and 190 nm and narrow size distribution. Yields of synthesis were 62–82% and can be further increased. Gelatin nanoparticles have good colloidal stability and withstand autoclaving. Moreover, they were non-toxic to human immune cells. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Funding: The reported study was funded by RFBR and Kaliningrad Oblast according to the research project № 19-415-393005 (preparation of fluorescence gelatin nanoparticles and study of their fluorescent properties), by RFBR research project 19-015-00408 (preparation of gelatin nanoparticles by the desolvation method), and by Ministry of Science and Higher Education of the Russian Federation within the framework of the Russian State Assignment under contract No. AAAA-А19-119112290010-7 (assessment of nanoparticles cytotoxicity).

Gelatin nanoparticles found numerous applications in drug delivery, bioimaging, immunotherapy, and vaccine development as well as in biotechnology and food science. Synthesis of gelatin nanoparticles is usually made by a two-step desolvation method, which, despite providing stable and homogeneous nanoparticles, has many limitations, namely complex procedure, low yields, and poor reproducibility of the first desolvation step. Herein, we present a modified one-step desolvation method, which enables the quick, simple, and reproducible synthesis of gelatin nanoparticles. Using the proposed method one can prepare gelatin nanoparticles from any type of gelatin with any bloom number, even with the lowest ones, which remains unattainable for the traditional two-step technique. The method relies on quick one-time addition of poor solvent (preferably isopropyl alcohol) to gelatin solution in the absence of stirring. We applied the modified desolvation method to synthesize nanoparticles from porcine, bovine, and fish with bloom values from 62 to 225 on the hundreds-of-milligram scale. Synthesized nanoparticles had average diameters between 130 and 190 nm and narrow size distribution. Yields of synthesis were 62-82% and can be further increased. Gelatin nanoparticles have good colloidal stability and withstand autoclaving. Moreover, they were non-toxic to human immune cells.

We studied the effects of pregnancy-specific β1-glycoprotein (PSG) on the replicative potential of naïve T cells (CD45RA

Background Pregnancy-specific β1-glycoproteins are capable of regulating innate and adaptive immunity, exerting predominantly suppressive effects. In this regard, they are of interest in terms of their pharmacological potential for the treatment of autoimmune diseases and post-transplant complications. The effect of these proteins on the main pro-inflammatory subpopulation of T lymphocytes, IL-17-producing helper T cells (Th17), has not been comprehensively studied. Therefore, the effects of the native pregnancy-specific β1-glycoprotein on the proliferation, Th17 polarization and cytokine profile of human CD4+ cells were assessed. Results Native human pregnancy-specific β1-glycoprotein (PSG) at а concentration of 100 μg/mL was shown to decrease the frequency of Th17 (RORγτ+) in CD4+ cell culture and to suppress the proliferation of these cells (RORγτ+Ki-67+), along with the proliferation of other cells (Ki-67+) (n = 11). A PSG concentration of 10 μg/mL showed similar effect, decreasing the frequency of Ki-67+ and RORγτ+Ki67+ cells. Using Luminex xMAP technology, it was shown that PSG decreased IL-4, IL-5, IL-8, IL-12, IL-13, IL-17, MIP-1β, IL-10, IFN-γ, TNF-α, G-CSF, and GM-CSF concentrations in Th17-polarized CD4+ cell cultures but did not affect IL-2, IL-7, and MCP-1 output. Conclusions In the experimental model used, PSG had а mainly suppressive effect on the Th17 polarization and cytokine profile of Th17-polarized CD4+ cell cultures. As Th17 activity and a pro-inflammatory cytokine background are unfavorable during pregnancy, the observed PSG effects may play a fetoprotective role in vivo.

The conditions for constructing an immunosorbent reagent for solid-phase NMR analysis were optimized. For this purpose, we increased the area of the sensitized portion of the membrane to fit the relaxometer coil size and added the agent sorption buffer. This provided the penetration of the anti-ligand molecules into the membrane thickness and their uniform distribution.

We studied the effect of the native (non-recombinant) alpha-fetoprotein (AFP) on differentiation, proliferation, and cytokine profile of activated helper T cells 17 (Th17). The object of the study was a culture of isolated by immunomagnetic separation helper T cells (CD4+), induced into the Th17 phenotype by using TCR-activator and proinflammatory cytokines (IL-1β and IL-6). AFP had not significant effect on the frequency of Th17 cells (ROR-γτ+) in the helper T cell culture, and did not affect proliferation of these cells, as measured by Ki-67 expression. Evaluation of the cytokine profile of culture supernatants by using the Luminex xMAP technology, revealed that AFP did not affect the levels of IL-4, IL-5, IL-7, IL-8, IL-10, IL-17, IFN-γ and TNF-α, but at concentrations of 50 IU/ml and 100 IU/ml it increased IL-2 production by activated helper T cells. At the same time, AFP suppressed the synthesis of G-CSF and GM-CSF (10 IU/ml), but stimulated the production of CCL4/MIP-1β (100 IU/ml) and CCL2/MCP-1 chemokines (10 IU/ml and 50 IU/ml).Issledovali vliianie nativnogo (ne rekombinantnogo) preparata al'fa-fetoproteina (AFP) na differentsirovku, proliferatsiiu i tsitokinovyĭ profil' aktivirovannykh T-khelperov 17 tipa (Th17). Ob"ektom issledovaniia sluzhili izolirovannye metodom immunomagnitnoĭ separatsii kul'tury “naivnykh” T-khelperov (CD4+), kotorye indutsirovali v fenotip Th17 pri pomoshchi TCR-aktivatora i provospalitel'nykh tsitokinov (IL-1β i IL-6). AFP ne okazyval dostovernogo vliianiia na uroven' Th17-kletok v kul'ture (CD4+ROR-γτ+), i ne vliial na proliferatsiiu étikh kletok, otsenivaemuiu po ékspressii Ki-67. Pri otsenke tsitokinovogo profilia po tekhnologii “Luminex xMAP” ustanovleno, chto AFP ne vliial na urovni IL-4, IL-5, IL-7, IL-8, IL-10, IL-17, IFN-γ i TNF-α, no v kontsentratsiiakh 50 ME/ml i 100 ME/ml povyshal produktsiiu IL-2 aktivirovannymi T-khelperami. V to zhe vremia AFP podavlial sintez G-CSF i GM-CSF (10 ME/ml), no stimuliroval produktsiiu khemokinov CCL4/MIP-1β (100 ME/ml) i CCL2/MCP-1 (10 ME/ml i 50 ME/ml).

We studied the effect of graphene oxide (GO) nanoparticles on differentiation of human myeloid suppressor cells (MDSC) in an in vitro system. Separated mononuclear cells of healthy donors were induced with cytokines (IL-6 and GM-CSF) into the MDSC phenotype (both polymorphonuclear (PMN-MDSC) and monocyte (M-MDSC) subsets of these cells were taken into account). Pegylated GO nanoparticles (GO-PEG; mean size 569±14 nm, PEG content ~20%) were used. GO-PEG in low concentrations (2.5 and 5 μg/ml) increased the percentage of MDSC in cultures, but reduced their content in high concentration (10 μg/ml). After exposure to GO-PEG (2.5 and 5 μg/ml), the MDSC content increased at the expense of M-MDSC, while the level of PMN-MDSC did not change. The decrease in MDSC levels after exposure to high doses of GO-PEG (10 μg/ml) was due to a decrease in PMN-MDSC. Thus, GO-PEG nanoparticles can oppositely regulate differentiation of MDSC by inhibiting or stimulating differentiation of these cells depending on the concentration.

The role of human chorionic gonadotropin (hCG) and its peptide fragments of β-subunit (LQGV, AQGV, VLPALP) in the regulation of indolеamine-2,3-dioxygenase (IDO) expression in human monocytes was studied in vitro. It is elucidated, that a high concentration of hCG (20 μg/ml) had an independent stimulatory eff ect on the expression of IDO in LPS -, and the IFN-γ – induced monocytes. Peptides AQGVand LQGV (20 μg/ml) also stimulated LPS-induced expression of IDO, but had no eff ect on IFN- γ-induced expression of the enzyme. At the same time, peptide VLPALP (20 μg/ml) provided the stimulatory eff ect only on the IFN-γ-induced expression of IDO. Simultaneously, peptides AQGV and VLPALP increased the production of IL-1β monocytes under LPS-stimulation, while hCG did not exert any independent eff ect. Generally, hCG and oligopeptides of β-hCG subunit enhance the expression of IDO by monocytes, which eventually contributes to peripheral immune tolerance development.

The effect of native α-fetoprotein (AFP) on the conversion of naïve T-helpers into central memory T-cells (TCM) and effector subpopulations of the preterminally differentiated (TEM) and terminally differentiated (TEMRA) memory T-cells was studied. AFP was found to prevent the conversion of naïve T-helpers into effector subpopulations of memory T cells (TEM and TEMRA) while reducing the total production of IL-4 and IFN-γ by the studied cell populations. The data reveal a new role of AFP in the immune tolerance formation during pregnancy.

The role of human chorionic gonadotropin (hCG) in the regulation of molecular genetics factors determining the functional activity of human naïve and memory T cells in vitro was studied. It was found that hCG (10 and 100IU/ml) inhibited CD28 and CD25 expression on the naïve T cells (CD45RA+) and CD25 expression on the memory T cells (CD45R0+). hCG didn't affect the CD71 proliferation marker expression in total. Nevertheless, hCG reduced the percentage of proliferating memory T cells with simultaneous suppression of CD71 expression on proliferating CD45R0+cells. In parallel, expression of U2af1l4, Gfi1, and hnRNPLL genes, which are Ptprc gene alternative splicing regulators was evaluated. It was established that hCG stimulated the expression of U2af1l4 and hnRNPLL genes, responsible for the assembly of CD45R0 in memory T cells, but reduced the expression of Gfi1 in these cells. In general, hCG promotes the differentiation of memory T cells by increasing of CD45R0 expression, but inhibits proliferation and CD25 expression which reflects their functional activity.

A dot immunoassay for simultaneous semiquantitative detection of IgG against tetanus toxoid (Ttx) and diphtheria toxoid (Dtx) and qualitative detection of anti-Bordetella pertussis IgGs in human blood serum using carbon nanoparticles functionalized with streptococcal protein G was developed. Inactivated B. pertussis cells in suspension form were used as an antigen in the immunoassay. Pertussis, tetanus, and diphtheria antigens were separately spotted onto nitrocellulose strips, and then the immunostrips were successively incubated with blood sera and a suspension of carbon nanoparticles. The immunostrips were then scanned with a flatbed scanner, and the images obtained were processed with ImageJ. One hundred fifty-five venous blood serum samples from children vaccinated with diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccine were tested in comparison with a conventional ELISA and agglutination test. The total time required for analysis of 32 serum samples was less than 3 h. Comparison between the results of the dot immunoassay and the corresponding ELISA/agglutination test revealed a high level of agreement (Cohen's kappa between 0.765 and 0.813). The lower limit of quantification was 0.06 IU/ml for anti-Ttx and anti-Dtx. The intra-assay coefficients of variation were less than 15% for anti-Ttx and anti-Dtx and less than 10% for anti-pertussis. The diagnostic sensitivity of detection of the antibody protection level was 93.5% for anti-Ttx [95% confidence interval (CI) 83.5-97.9%], 92.4% for anti-Dtx (95% CI 80.9297.5%), and 90.2% for anti-pertussis (95% CI 75.9-96.8%). The diagnostic specificity was 90.9% for anti-Ttx (95% CI 57.1-99.5%), 85% for anti-Dtx (95% CI 61.1-96.0%), and 89.3% for anti-pertussis (95%CI 80.8-94.5%). The dot immunoassay developed does not require expensive reading equipment, and allows detection of antibodies against three antigens in a single analysis. The immunostrips can be stored for a long time without changes in the coloration of the spots. Graphical Abstract The assay procedure. BC Bordetella pertussis cell suspension, CNP carbon nanoparticle, Dtx diphtheria toxoid, Ttx tetanus toxoid.

A solid phase NMR-based sandwich immunoassay for the prostate-specific antigen (PSA) is presented. Carbon-encapsulated iron nanoparticles were functionalized with bovine serum albumin, coupled to monoclonal antibodies, and then used as magnetic labels. A nitrocellulose membrane with 8-μm pores was coated with capture antibodies and subsequently incubated with a serum sample and a suspension of the nanoconjugate. Test strips were placed in a portable homemade NMR relaxometer. Magnetic nanoparticles attached to nitrocellulose decrease the T2 relaxation time of the water protons located inside the pores of the membrane. Thus, T2 is inversely proportional to the concentration of the antigen (PSA) in the sample. The assay can be performed within 4 h. The detection limit is 0.44 ng mL−1. Kallikrein 2, human chorionic gonadotropin, and α-fetoprotein do not interfere.

The surface functionalization of magnetic nanoparticles improves their physicochemical properties and applicability in biomedicine. Natural polymers, including proteins, are prospective coatings capable of increasing the stability, biocompatibility, and transverse relaxivity (r2) of magnetic nanoparticles. In this work, we functionalized the nanoclusters of carbon-coated iron nanoparticles with four proteins: bovine serum albumin, casein, and gelatins A and B, and we conducted a comprehensive comparative study of their properties essential to applications in biosensing. First, we examined the influence of environmental parameters on the size of prepared nanoclusters and synthesized protein-coated nanoclusters with a tunable size. Second, we showed that protein coating does not significantly influence the r2 relaxivity of clustered nanoparticles, however, the uniform distribution of individual nanoparticles inside the protein coating facilitates increased relaxivity. Third, we demonstrated the applicability of the obtained nanoclusters in biosensing by the development of a nuclear-magnetic-resonance-based immunoassay for the quantification of antibodies against tetanus toxoid. Fourth, the protein coronas of nanoclusters were studied using SDS-PAGE and Bradford protein assay. Finally, we compared the colloidal stability at various pH values and ionic strengths and in relevant complex media (i.e., blood serum, plasma, milk, juice, beer, and red wine), as well as the heat stability, resistance to proteolytic digestion, and shelf-life of protein-coated nanoclusters.

Research paper on sunthesis of protein nanoparticlesAbstractThe desolvation technique is one of the most popular methods for preparing protein nanoparticles for medicine, biotechnology, and food applications. We fabricated 11 batches of BSA nanoparticles and 2 batches of gelatin nanoparticles by desolvation method. BSA nanoparticles from 2 batches were cross-linked by heating at +70 °C for 2 h; other nanoparticles were stabilized by glutaraldehyde. We compared several analytical approaches to measuring their concentration: gravimetric analysis, bicinchoninic acid assay, Bradford assay, and alkaline hydrolysis combined with UV spectroscopy. We revealed that the cross-linking degree and method of cross-linking affect both Bradford and BCA assay. Direct measurement of protein concentration in the suspension of purified nanoparticles by dye-binding assays can lead to significant (up to 50-60%) underestimation of nanoparticle concentration. Quantification of non-desolvated protein (indirect method) is affected by the presence of small nanoparticles in supernatants and can be inaccurate when the yield of desolvation is low. The reaction of cross-linker with protein changes UV absorbance of the latter. Therefore pure protein solution is an inappropriate calibrator when applying UV spectroscopy for the determination of nanoparticle concentration. Our recommendation is to determine the concentration of protein nanoparticles by at least two different methods, including gravimetric analysis.

The goal of our study was to compare the following two methods of assessment of pertussis post-vaccination immunity: bacterial agglutination test and pertussis toxin enzyme-linked immunosorbent assay (ELISA).The study was carried out in Perm Region, Russia. We measured pertussis immunity using two serological methods: ELISA of IgG to pertussis toxin (PT) and the agglutination test (AT) among 135 children, in the age range from 2 months to 17 years old. The immunization schedule included four doses of DTwP: at 3, 4.5 and 6 months of age and a booster at 18 months. All participants were divided into six age groups.The percentage of samples with IgG level less than the detection limit in vaccinated children was 52.2%. The total seropositivity rate (the percent of children with agglutinin titres ≥1:160) in vaccinated children was 47.8%. Only a weak association was observed between agglutinin and anti-PT IgG titres (R = .3). Neither the primary nor the booster vaccination with DTwP influenced the IgG levels in children. Agglutinin titres significantly increased after vaccination and declined 5 years after the booster dose. Significant growth of IgG concentration was observed in 11-year-olds, indicating the presence of B. pertussis circulation in the childhood population.Based on the obtained results and the results of other authors, we summarize that anti-PT ELISA should be carefully used to assess the population immunity to pertussis. Currently, there is neither a serological test that accurately determines the protection against pertussis nor a distinctive criterion of protection that can be applied in seroepidemiological studies.

The effects of chorionic gonadotropin (hCG) on the expression of the hTERT gene in combination with the conversion of the phenotype of naive T-cells and T-cells of immune memory in vitro were studied. hCG inhibited expression of hTERT mRNA in naive T-cells (CD45RA+) and immune memory T cells (CD45RO+), causing a decrease in the replicative potential of the cells. The presence of hCG in the culture led to the conversion of the phenotype of T-lymphocytes. hCG reduced the number of proliferating T-cells of immune memory, estimated by phenotypic signs by differential gating. hCG (10 IU/ml and 100 IU/ml) inhibited expression of CD25 by the studied populations, but did not modulate expression of the CD71 proliferation marker. Thus, hCG inhibited the functional activity of naive T-cells and T-cells of immune memory, which, in the context of pregnancy, can contribute to the formation of immune tolerance to the semi-allogenic fetus.Izuchali éffekty khorionicheskogo gonadotropina (KhG) na ékspressiiu gena hTERT v sochetanii s konversieĭ fenotipa naivnykh T-kletok i T-kletok immunnoĭ pamiati v sisteme in vitro. Ustanovleno, chto KhG ugnetal ékspressiiu mRNK gena hTERT v naivnykh T-kletkakh (CD45RA+) i T-kletkakh immunnoĭ pamiati (CD45RO+), chto privodilo k snizheniiu replikativnogo potentsiala kletok. Prisutstvie KhG v kul'ture privodilo k konversii fenotipa T-limfotsitov. KhG snizhal chislo proliferiruiushchikh T-kletok immunnoĭ pamiati, otsenivaemykh po fenotipicheskim priznakam metodom differentsial'nogo geĭtirovaniia, podavlial ékspressiiu markera aktivatsii CD25 issleduemymi populiatsiiami, ne vliiaia na ékspressiiu CD71, assotsiirovannogo s proliferatsieĭ. Takim obrazom, KhG snizhal replikativnyĭ potentsial i podavlial aktivatsiiu T-kletok i T-kletok immunnoĭ pamiati, chto v kontekste beremennosti mozhet vnosit' vklad v formirovanie immunnoĭ tolerantnosti k poluallogennomu émbrionu.

Paper description: Pregnancy-specific β-1-glycoproteins (PSGs) are capable of regulating innate and adaptive immunity. As fetal antigens circulate in pregnant women blood, it is of particular interest to reveal the effects of PSGs on memory T cell differentiation in the context of maternal-fetal tolerance development. We found that PSG reduces the frequencies of CD4 + CD45RA + effector memory (TEMRA) and CD45RA + CD45R0 + T-cells, while inhibiting IL-4 and IFN-γ production by total helper T-cells in vitro . As TEMRA-cells exhibit cytotoxic activity that is unfavorable during pregnancy, the revealed PSG effects might play a fetoprotective role in vivo . Abstract: Pregnancy-specific β-1-glycoproteins (PSGs) are capable of regulating innate and adaptive immunity. As fetal antigens circulate in the blood of pregnant women, it is of particular interest to reveal the effects of PSGs on the differentiation of memory T cells in the context of maternal-fetal tolerance formation. We studied if, native PSG preparation affects helper T-cell proliferation, the frequencies of CD4 + CD45R0 + , CD4 + CD45RA + , CD4 + CD45RA + CD45R0 + cells, naive CD45RA + CD45R0 - CD62L + cells (NAIVE), central memory CD45RA - CD45R0 + CD62L + cells (TCM) and effector memory helper T cells (CD45RA - CD45R0 + CD62L - (TEM) and CD45RA + CD45R0 - CD62L - cells (TEMRA), together with IL-4 and IFN-γ production by all СD4 + T cells. The suppressive effect of PSG on helper T-cell proliferation was established. It was found that PSG does not influence the frequencies of CD45RA + and CD45R0 + cells, while it decreased the percentage of CD45RA + CD45R0 + cells. PSG increased the percentage of NAIVE cells in culture, and prevented the conversion of these cells into TEMRA, without affecting the levels of TCM and TEM. In addition, PSG lowered the amount of IL-4 and IFN-γ in the supernatants of helper T-cell cultures. As TEMRA exhibit cytotoxic activity that is unfavorable during pregnancy, the revealed PSG effects may play a fetoprotective role in vivo . https://doi.org/10.2298/ABS190122019T Received: January 22, 2019; Revised: March 15, 2019; Accepted: March 21, 2019; Published online: March 25, 2019 How to cite this article: Timganova V, Bochkova M, Khramtsov P, Kochurova S, Rayev M, Zamorina S. Effects of pregnancy-specific β-1-glycoprotein on the helper T-cell response. Arch Biol Sci. 2019;71(2):369-78.

Conjugates of carbon nanoparticles and aptamers have great potential in many areas of biomedicine. In order to be implemented in practice, such conjugates should keep their properties throughout long storage period in commonly available conditions. In this work, we prepared conjugates of carbon nanoparticles (CNP) with DNA aptamers using streptavidin-biotin reaction. Obtained conjugates possess superior stability and kept their physical-chemical and functional properties during 30 days at +4 °C and -20 °C. Proposed approach to conjugation allows loading of about 100-120 pM of biotinylated aptamer per 1 mg of streptavidin-coated CNP (CNP-Str). Aptamer-functionalized CNP-Str have zeta potential of -34 mV at pH 7, mean diameter of 168-177 nm, and polydispersity index of 0.080-0.140. High reproducibility of functionalization was confirmed by preparation of several batches of CNP-aptamer with the same size distribution and aptamer loading using independently synthesized parent CNP-Str nanoparticles. Stability of CNP-aptamer conjugates was significantly enhanced by postsynthesis addition of EDTA that prevents nuclease degradation of immobilized aptamers. Obtained nanoparticles were stable at pH ranging from 6 to 10. Optical properties of CNP-aptamer nanoparticles were also studied and their ability to quench fluorescence via Forster resonance energy transfer was shown. Taking into account properties of CNP-aptamer conjugates, we suppose they may be used in both homo- and heterogeneous colorimetric, fluorescent, and aggregation-based assays.